Aconitum alkaloids are versatile in chemical structures and are well known for their bioactivity and toxicity. Cases of analogs with closely similar structures or positional isomers are widespread in herbs of the Aconitum genus. It is still challenging to rapidly identify unknown compounds via mass spectrometry, especially positional isomers. Herein, to profile the alkaloids of Aconitum stapfianum that possess bioactivity against intoxication by the lateral root of Aconitum carmichaelii (Fuzi), a strategy was developed by carefully determining the fragmentation pathways of authentic standards. A series of rules was summarized and involved charge site effects, hydrogen bonding effects, competitive channels between charge-remote reactions and charge migration reactions, and fragment patterns exhibiting a diamond shape. Accordingly, a total of 124 alkaloids in A. stapfianum were tentatively characterized, including 85 potential new compounds and 24 sets of isomers. On the other hand, to explore the material basis of detoxification, a chemical constituent comparison was made between A. stapfianum and Fuzi, and principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed to identify markers that were different between the two . In total, 25 characteristic markers were identified to discriminate between these two herbal medicines, of which 14 compounds were specific for A. stapfianum and most of them were characteristic for a para-substituted benzoic acid ester at C-14. (c) 2021 Elsevier B.V. All rights reserved.